title: Development of a high-throughput cell-based reporter assay for screening of JAK3 inhibitors.
abstract: JAK3 is an ideal target for the treatment of immune-related diseases and the prevention of organ allograft rejection. Several JAK3 inhibitors have been identified by biochemical enzymatic assays, but the majority display significant off-target effects on JAK2. Therefore, there is a need to develop new experimental approaches to identify compounds that specifically inhibit JAK3. Here, we show that in 32D/IL-2Rβ cells, STAT5 becomes phosphorylated by an IL-3/JAK2- or IL-2/JAK3-dependent pathway. Importantly, the selective JAK3 inhibitor CP-690,550 blocked the phosphorylation and the nuclear translocation of STAT5 following treatment of cells with IL-2 but not with IL-3. In an attempt to use the cells for large-scale chemical screens to identify JAK3 inhibitors, we established a cell line, 32D/IL-2Rβ/6xSTAT5, stably expressing a STAT5 reporter gene. Treatment of this cell line with IL-2 or IL-3 dramatically increased the reporter activity in a high-throughput format. As expected, CP-690,550 selectively inhibited the activity of the 6xSTAT5 reporter following treatment with IL-2. By contrast, the pan-JAK inhibitor curcumin inhibited the activity of this reporter following treatment with either IL-2 or IL-3. Thus, this study indicates that the STAT5 reporter cell line can be used as an efficacious cellular model for chemical screens to identify selective JAK3 inhibitors.
authors.affiliation: Department of Pediatrics, Division of Hematology/Oncology, New York Medical College, Valhalla, NY 10595, USA.
authors.forename: Chang-Hong
authors.initials: CH
authors.lastname: Yin
authors.forename: Erika A
authors.initials: EA
authors.lastname: Bach
authors.forename: Gyeong-Hun
authors.initials: GH
authors.lastname: Baeg
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D004353:Drug Evaluation, Preclinical; D004789:Enzyme Activation / Q000187:drug effects*; D017930:Genes, Reporter / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000379:methods*; D007376:Interleukin-2 / Q000378:metabolism; D007377:Interleukin-3 / Q000378:metabolism; D053616:Janus Kinase 3 / Q000037:antagonists & inhibitors*; D051379:Mice; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D050799:STAT5 Transcription Factor / Q000235:genetics / Q000378:metabolism
chemical_list: D007376:Interleukin-2; D007377:Interleukin-3; D047428:Protein Kinase Inhibitors; D050799:STAT5 Transcription Factor; D053616:Janus Kinase 3